Mesothelin, immunogenic peptides derived therefrom, and compositions comprising mesothelin, or immunogenic peptides thereof

ABSTRACT

This invention relates to the discovery of a differentiation antigen termed mesothelin which is associated with mesotheliomas and ovarian cancers. Mesothelin is about 69 kD in its full-length form. The invention includes uses for the amino acid and nucleic acid sequences for mesothelin, recombinant cells expressing it, methods for targeting and/or inhibiting the growth of cells bearing mesothelin, methods for detecting the antigen and its expression level as an indication of the presence of tumor cells, and kits for such detection.

This application is a divisional of U.S. application Ser. No.09/215,035, filed Dec. 17, 1998, now U.S. Pat. No. 6,153,430 which is adivisional of U.S. application Ser. No. 08/776,271, filed Jan. 12, 1998,now U.S. Pat. No. 6,083,502 which is a national stage filing under 35U.S.C. § 371 of PCT/US97/00224, filed Jan. 3, 1997, which claimspriority from U.S. Provisional Application No. 60/010,166, filed Jan. 5,1996, all of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the identification of a specific antigen foundon tumor cells, particularly mesotheliomas and ovarian tumor cells and,inter alia, methods and compositions for targeting cells bearing theantigen.

BACKGROUND OF THE INVENTION

Monoclonal antibodies are currently being used to diagnose and treatcancer (Mach, J., et al., Current Opinion Immunol. B, 685-693 (1991);Grossbard, M. L., et al., Blood 80 (4):863-878 (1992)). To be useful fortherapy, the antibody should recognize an antigen that is present inlarge amounts on the cancer cells and in negligible amounts in normalcells. Alternatively the antigen can be present in substantial amountson normal cells, if the normal cells are not components of an essentialorgan. This approach has been useful in developing new treatments forleukemias and lymphomas. Several differentiation antigens have beenidentified on lymphomas and leukemias which are good targets forimmunotherapy, because they are not present on the stem cells which giverise to differentiated lymphocytes (Grossbard, M. L., et al., Blood 80(4):863-878 (1992)). Thus, normal lymphocytes that are killed byimmunotherapy can be regenerated. Some examples of lymphocyte antigensof this type are CD19, CD22, CD25 and CD30 (Grossbard, M. L., et al.,Blood 80 (4):863-878 (1992); Engert, A., et al., Cancer Research 50,84-88 (1990)). Clearly, it would be very useful to have antibodies thatrecognize differentiation antigens on solid tumors, but only a smallnumber of these are available. One reason contributing to the paucity ofsuch antibodies is that efforts to identify differentiation antigens onvarious types of epithelial cells have been relatively modest comparedwith the intense efforts made to identify differentiation antigens oncells of the hematopoietic system.

Ovarian cancer represents one of the diseases which could be treated byimmunotherapy, because the ovaries are always removed during surgery forthis disease and reactivity with normal ovarian tissue is not a problem.Several antibodies that recognize differentiation antigens on ovariancancer cells have been generated. One of these is OC125 that recognizesthe CA125 antigen (Bast, R., et al., N. Eng. J. Med. 309, 883-887(1983)). CA125 is a high molecular weight glycoprotein that is shed byovarian cancer cells and has been useful in the diagnosis of ovariancancer. However, antibodies to CA125 are not useful for immunotherapybecause the CA125 antigen is shed into the blood stream (Bast, R., etal., N. Eng. J. Med. 309, 883-887 (1983)). Another is MOV18 whichrecognizes the folate binding protein. This protein is abundant inovarian cancers as well as in some other tumors. Unfortunately, thisprotein is also abundantly expressed in kidney (Campbell, I. G., et al.,Cancer Res. 51, 5329-5338 (1991)). An antibody we previously isolatedand termed MAb K1 reacts with many ovarian cancers and manymesotheliomas. Like OC125, the antibody also reacts with normalmesothelial cells, but it does not react with other cell types exceptfor weak reactivity with some cells in the trachea (Chang, K., et al.,Int. J. Cancer 50, 373-381 (1992); Chang, K., et al., Cancer Res. 52,181-186 (1992), see also U.S. Pat. No. 5,320,956). The antigenrecognized by MAb K1 appears to be a differentiation antigen present onmesothelium and is expressed on cancers derived from mesothelium such asepithelioid type mesotheliomas as well as on most ovarian cancers. Thusimmunotherapy directed at the CAK1 antigen should take into account thepotential risk of damaging normal mesothelial cells and perhaps cells ofthe trachea (Chang, K., et al., Int. J. Cancer 50, 373-381 (1992);Chang, K., et al., Cancer Res. 52, 181-186 (1992); Chang, K., et al.,Int. J. Cancer 51, 548-554 (1992); Chang, K., et al., Am. J. Surg.Pathol. 16, 259-268 (1992)).

Using the ovarian cancer cell line OVCAR-3 as well as HeLa cells, theantigen has been shown to be an approximately 40 kD glycoprotein that isattached to the cell surface by phosphatidylinositol. The protein isreleased when cells are treated with phosphotidylinositol specificphospholipase C (Chang, K., et al., Cancer Res. 52, 181-186 (1992)). Wehad previously attempted to clone a cDNA encoding the CAK1 antigen butinstead cloned cDNAs encoding two different intracellular proteins whichalso react with MAb K1 (Chang, K., and Pastan, I., Int. J. Cancer 57,90-97 (1994)). Neither of these is the cell surface membrane antigenrecognized by MAb K1.

SUMMARY OF THE INVENTION

The present invention provides uses for isolated polypeptides comprisingat least 10 contiguous amino acids from the polypeptide sequence of SEQID NO: 2, wherein the polypeptide binds to antisera raised against thefull-length polypeptide of SEQ ID NO: 2 as an immunogen, which has beenfully immunoadsorbed with a 40 kD polypeptide attached to the cellsurface of OVCAR-3 and HeLa cells (the K1 antigen). Full-lengthpolypeptides of the invention are typically about 69 kD in size,although they are larger when glycosylated or incorporated into aconstruct such as an eukaryotic expression vector. The polypeptides ofthe present invention may be present in several forms, includingisolated naturally occurring endoproteolytic polypeptides, recombinantlyproduced polypeptides, and as portions of recombinant polypeptides suchas fusion proteins.

The present invention also provides uses for isolated nucleic acidswhich encode the polypeptides described above. Exemplary nucleic acidsinclude those described in SEQ ID NO: 1. In preferred embodiments, thenucleic acid is part of a recombinant vector such as a plasmid or virusor may be used as a probe to detect for the antigen. In preferredembodiments, the nucleic acid selectively hybridizes to the nucleic acidof SEQ ID NO: 1. The nucleic acid sequence may encode, e.g., amesothelin polypeptide with complete sequence identity to a naturallyoccurring mesothelin protein. The nucleic acid may also encode amesothelin polypeptide which is not identical to a naturally occurringmesothelin polypeptide, such as a fusion protein, or a geneticallyengineered mutant mesothelin protein which retains the bases criticalfor protein function or immunogenicity as described herein.

Recombinant cells which comprise a nucleic acid of the present inventionare also provided, including eukaryotic and prokaryotic cells. Thepresent invention also provides antibodies which bind specifically tothe polypeptides of the present invention.

The invention further provides methods for targeting and/or inhibitingthe growth of cells bearing mesothelin; methods for detecting theantigen and its expression level as an indication of the presence oftumor cells; and kits for such detection.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The term “antibody” as used herein, includes various forms of modifiedor altered antibodies, such as an intact immunoglobulin, variousfragments such as an Fv fragment, an Fv fragment containing only thelight and heavy chain variable regions, an Fv fragment linked by adisulfide bond (Brinkmann, et al. Proc. Natl. Acad. Sci. USA, 90:547-551 (1993)), an Fab or (Fab)′₂ fragment containing the variableregions and parts of the constant regions, a single-chain antibody andthe like (Bird et al., Science 242: 424-426 (1988); Huston et al., Proc.Nat. Acad. Sci. USA 85: 5879-5883 (1988)). The antibody may be of animal(especially mouse or rat) or human origin or may be chimeric (Morrisonet al., Proc Nat. Acad. Sci. USA 81: 6851-6855 (1984)) or humanized(Jones et al., Nature 321: 522-525 (1986), and published UK patentapplication #8707252).

The term “immunoassay” is an assay that utilizes an antibody tospecifically bind an analyte or antigen. The immunoassay ischaracterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the analyte.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial which is substantially or essentially free from componentswhich normally accompany it as found in its native state.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides.

The term “nucleic acid probe” refers to a molecule which binds to aspecific sequence or subsequence of a nucleic acid. A probe ispreferably a nucleic acid which binds through complementary base pairingto the full sequence or to a subsequence of a target nucleic acid. Itwill be understood by one of skill in the art that probes may bindtarget sequences lacking complete complementarity with the probesequence depending upon the stringency of the hybridization conditions.The probes are preferably directly labelled as with isotopes,chromophores, lumiphores, chromogens, or indirectly labelled such aswith biotin to which a streptavidin complex may later bind. By assayingfor the presence or absence of the probe, one can detect the presence orabsence of the select sequence or subsequence.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “recombinant” when used with reference to a cell indicates thatthe cell encodes a DNA whose origin is exogenous to the cell-type. Thus,for example, recombinant cells express genes that are not found withinthe native (non-recombinant) form of the cell.

The term “identical” in the context of two nucleic acids or polypeptidesequences refers to the residues in the two sequences which are the samewhen aligned for maximum correspondence. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homologyalignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson and Lipman Proc. Natl.Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by inspection.

The term “substantial identity” or “substantial similarity” in thecontext of a polypeptide indicates that a polypeptide comprises asequence with at least 70% sequence identity to a reference sequence, orpreferably 80%, or more preferably 85% sequence identity to thereference sequence, or most preferably 90% identity over a comparisonwindow of about 10-20 amino acid residues. An indication that twopolypeptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a polypeptide is substantially identical to a secondpolypeptide, for example, where the two peptides differ only by aconservative substitution.

An indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide which the first nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid.

Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. However, nucleic acids which do not hybridizeto each other under stringent conditions are still substantiallyidentical if the polypeptides which they encode are substantiallyidentical. This occurs, e.g., when a copy of a nucleic acid is createdusing the maximum codon degeneracy permitted by the genetic code.

The phrases “specifically binds to a protein” or “specificallyhybridizes to” or “specifically immunoreactive with”, when referring toan antibody refers to a binding reaction which is determinative of thepresence of the protein in the presence of a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind preferentially to a particularprotein and do not bind in a significant amount to other proteinspresent in the sample. Specific binding to a protein under suchconditions requires an antibody that is selected for its specificity fora particular protein. A variety of immunoassay formats may be used toselect antibodies specifically immunoreactive with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York, for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Nucleotide sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of the CAK1-9 cDNA. The nucleotide sequence(upper line) and the deduced amino acid sequence (lower line) of theCAK1 cDNA is listed with nucleotide numbers at left. The translation ofCAK1 starts at nucleotides 100-102 (ATG) and terminates at 1986-88(TGA). The putative signal peptide is underlined and a typicalhydrophobic sequence for GPI anchorage is double-underlined. A likelyfurin cleavage site RPRFRR is underlined and the cleavage site shown byan arrow. There are four potential N-linked glycosylation sites (in boldletters). A variant polyadenylation signal (AGTAAA) is present 22 basepairs upstream from the polyadenylation tail. The original p6-1 cDNAsequence spans nucleotides 721 to 213.

FIG. 2: Different forms of the CAK1 tumor antigen. S.P.=putative signalpeptide; H.P.=GPI anchorage dependent hydrophobic peptide;CHO=carbohydrates; M=membrane, AA=amino acids.

DETAILED DESCRIPTION

This invention relates to the discovery of an antigen, referred toherein mesothelin, found on mesothelium, mesotheliomas, ovarian cancercells and some squamous cell carcinomas. Previously, an antibodydesignated monoclonal antibody K1 was described which reacts with anantigen found on OVCAR-3 cells (from a human ovarian tumor cell line)having a molecular weight of 40 kD (kilodaltons). e.g. U.S. Pat. No.5,320,956. The antigen described and claimed here was unexpectedlyobtained during an attempt to clone and sequence the K1 antigen.Mesothelin in its full-length form has an apparent molecular weight ofabout 69 kD and appears to be the precursor protein for the 40 kD K1antigen. The K1 antigen itself proved difficult to clone and our firstattempts resulted in the cloning of two different intracellular proteinsas mentioned above (see Chang & Pastan, Int. J. Cancer, supra). Thoughthe existence of the K1 antigen was known, its cDNA was not routine toclone. First, we were not able to obtain sufficient amounts of it toclone. The methods used here were more laborious, but successful becauseunbeknownst to us the K1 antigen was derived from a larger molecule thatwe did not know existed. The DNA sequence and corresponding amino acidsequence for full-length mesothelin are set out in FIG. 1 and in SEQ IDNOS: 1 and 2, respectively.

Reference to mesothelin herein refers to both the isolated full-lengthpolypeptide and isolated polypeptide fragments of at least 10 contiguousamino acids from the full-length sequence wherein the fragment binds toantisera raised against the full-length polypeptide, which has beenfully immunosorbed with the 40 kD K1 antigen.

Mesothelin, as described here represents an antigen which is found onmesothelium, mesotheliomas, ovarian cancers and some squamous cellcarcinomas. We have designated this antigen mesothelin to reflect itspresence on mesothelial cells. The full-length cDNA for mesothelin is2138 bp in length and contains an open reading frame of 1884 bp. Theprotein it encodes contains 628 amino acids with a calculated molecularweight of about 69000 daltons in its full-length form.

The protein contains four potential N-linked glycosylation sites N-X-Sor N-X-T that are shown in bold letters in FIG. 1. A typical signalsequence is not present at the amino terminus. However, a shorthydrophobic segment is located 15 amino acids from the first methionine(FIG. 1). This sequence might function as a signal sequence for membraneinsertion, because the protein is found on the cell surface and isinserted into microsomes during cell free translation. Also present is aputative proteolytic processing site, RPRFRR, beginning at amino acid293 (FIG. 1). This site is recognized by furin, a protease important inthe processing of several membrane proteins as well as in the activationof Pseudomonas and diphtheria toxins (Chiron, M. F., et al., J.B.C.269(27):18169-18176 (1994)).

The 40 kD form (“K1”) appears to be derived from a 69 kD precursor byseveral processing steps. These are summarized in FIG. 2. Initially,mesothelin is made as a 69 kD polypeptide with a hydrophobic tail whichis probably removed and replaced by phosphatidylinositol (Chang, K., etal., Cancer Res. 52, 181-186 (1992)). After glycosylation at one or moreof its four putative N-linked glycosylation sites, it is cleaved by aprotease to yield higher molecular weight forms, the 40 kD fragment (ordoublet) found on the surface of OVCAR-3 cells and a smaller (˜31 kD)fragment. The latter could be released into the medium and/or furtherdegraded. We found that the amino terminal fragment was detected in themedium of OVCAR-3 cells.

Mesothelin is one of many proteins and glycoproteins that are attachedto the cell surface by phosphatidylinositol. Several functions have beenascribed to these molecules. Some are receptors involved in cellsignaling; others are involved in cellular recognition and/or adhesion(Dustin, M. L., et al., Nature 329, 846-848 (1987); Stiernberg, J., etal., J. Immunol. 38, 3877-3884 (1987)). GPI linked proteins may interactwith tyrosine kinases (Stefanova, I., et al., Science 254, 1016-1019(1991); Pandey, A., et al., Science 268, 567-569 (1995)). Antibodies tomesothelin would be useful in inhibiting the spread or implantation ofovarian cancer cells into the peritoneal wall that sometimes occurs, forexample, during ovarian cancer surgery. Without intending to be bound bytheory, it is our belief that mesothelin is likely responsible for theadhesion and implantation of ovarian carcinoma cells that frequentlyoccurs throughout the peritoneal cavity or the adhesion of tumor cellsin the thoracic cavity. Mesothelin plays a role in adhesion sincemesothelin transfectants are more slowly removed from culture dishesthan non-transfected cells. Mesothelial cells are extremely flat andregulate the traffic of molecules and cells in and out of the peritonealor thoracic cavity.

Mesothelin is very abundant in normal mesothelial cells from whichmalignant mesotheliomas and ovarian cystadenocarcinomas are derived.These two types of tumors share a unique biological characteristic thatdistinguishes them from other solid tumors. In the early stages, bothtypes of tumors spread aggressively throughout the peritoneal (orthoracic) cavity and invade locally but do not metastasize distallythrough lymphatics or the blood stream. In fact, many patients succumbto their cancer before distant metastases develop. Mesothelin likely hasa role in this process, since cells overexpressing mesothelin havealtered adhesive properties and mesothelin expression is diminished inpoorly differentiated ovarian cancers (Chang, K., et al., Int. J. Cancer51, 548-554 (1992); Chang, K., et al., Am. J. Surg. Pathol. 16, 259-268(1992)). Implantation of ovarian cancer cells through a strong adhesionmechanism may be the first step towards local invasion and distalmetastasis. Thus, blocking ovarian cancer implantation will preventinvasion and metastasis as well as proliferation of the cancer cells andlead cancer cells to apoptosis and the like.

I. Detection for Mesothelin

The detection of mesothelin is useful as an indicator of the presence oftumor cells, particularly ovarian tumor cells or mesotheliomas. If foundin serum it can be a factor indicating the presence of residual cancercells. Tumor tissues contain various proteases which may be responsiblefor the cleavage of mesothelin. The amount of N-terminal fragment ofmesothelin present in blood or ascitic fluid can reflect the number ofresidual tumor cells present. The serological detection of mesothelinmay serve as a novel indicator for monitoring the process of disease.The basic principle for detection of the mesothelin proteins is todetect the protein using specific ligands that bind to mesothelin butnot to other proteins or nucleic acids in a normal human cell or itsenvirons. The ligands can be either nucleic acid or antibodies. Theligands can be naturally occurring or genetically or physically modifiedsuch as non-natural or antibody derivatives, i.e. FAB, or chimericantibodies.

A. Sample Collection and Processing

Mesothelin is preferably quantified in a biological sample, such as aserum, cell, or a tissue sample derived from a patient. In a preferredembodiment, mesothelin is quantified in samples of serum, mesothelialcells, cervical tissue or ovarian tissue with reference to a standardprepared from recombinant mesothelin.

The sample may be pretreated as necessary by dilution in an appropriatebuffer solution or concentrated, if desired depending upon the assaybeing used. Any of a number of standard aqueous buffer solutions,employing one of a variety of buffers, such as phosphate, Tris, or thelike, at physiological pH can be used.

B. Quantification of Mesothelin Peptides.

Mesothelin peptides may be detected and quantified by any of a number ofmeans well known to those of skill in the art. These include analyticbiochemical methods such as electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, and variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like.

C. General Techniques—Nucleic Acid Detection

Accepted means for conducting hybridization assays for detection areknown and general overviews of the technology can be had from a reviewof: Nucleic Acid Hybridization: A Practical Approach, Ed. Hames, B. D.and Higgins, S. J., IRL Press, 1985; Hybridization of Nucleic AcidsImmobilized on Solid Supports, Meinkoth, J. and Wahl, G.; AnalyticalBiochemistry, Vol 238, 267-284, 1984 and Innis et al., PCR Protocols,supra, all of which are incorporated by reference herein.

If PCR is used, for example, primers are designed to target a specificportion of the nucleic acid of the targeted agent. Preferably theprimers are about 14 to about 24 nucleotides in length. From thesequence information provided herein, those of skill in the art will beable to select appropriate specific primers.

Target specific probes may be used in the nucleic acid hybridizationdiagnostic assays for mesothelin. The probes are specific for orcomplementary to the target of interest. For example, probes to one ofthe nucleic acid sequences in the open reading frame for mesothelinwould be effective. For precise allelic differentiation, the probesshould be about 14-nucleotides long and preferably about 20-30nucleotides. For more general detection, nucleic acid probes are about50 to about 1000 nucleotides, most preferably about 200 to about 400nucleotides.

The detection of the mesothelin polypeptides and other aspects of thepresent invention may make use of techniques such as PCR, TAS, 3SR, QBamplification and cloning, to amplify a nucleic acid in a biologicalsample which encodes a mesothelin polypeptide for detection or for,inter alia, the production of probes and primer tools for detection.

The presence of mesothelin nucleic acid in a biological sample such as,for example, serum or tissue suspected to contain tumor cells, isuseful, e.g., as a probe to assess the presence of mesothelin andsubsequently provide evidence indicative of tumor cells.

The nucleic acids of the present invention are cloned, or amplified byin vitro methods, such as the polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR) andthe Qβ replicase amplification system (QB). A wide variety of cloningand in vitro amplification methodologies are well-known to persons ofskill. Examples of these techniques and instructions sufficient todirect persons of skill through many cloning exercises are found inBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.)Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,(Sambrook et al.); Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement) (Ausubel); Cashion et al., U.S. Pat. No. 5,017,478; andCarr, European Patent No. 0,246,864. Examples of techniques sufficientto direct persons of skill through in vitro amplification methods arefound in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987)U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (Innis); Arnhein & Levinson (Oct. 1, 1990) C&EN 36-47; TheJournal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci.USA 87, 1874; Lomell et al. (1989) J. Clin. Chem. 35, 1826; Landegren etal., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8,291-294; Wu and Wallace, (1989) Gene 4, 560; and Barringer et al. (1990)Gene 89, 117.

It will be readily understood by those of skill in the art and it isintended here, that when reference is made to particular sequencelistings, such as SEQ ID NOS: 1 and 2, such reference includes sequenceswhich substantially correspond to its complementary sequence and thosedescribed including allowances for minor sequencing errors, single basechanges, deletions, substitutions and the like, such that any suchsequence variation corresponds to the nucleic acid sequence to which therelevant sequence listing relates.

D. Antibodies to Mesothelin and Antibody-Ligand Binding Assays

Antibodies (or antisera) are raised to the polypeptides of the presentinvention, including individual fragments thereof, both in theirnaturally occurring (full-length) forms and in recombinant forms.Additionally, antibodies are raised to these polypeptides in eithertheir native configurations or in non-native configurations.Anti-idiotypic antibodies can also be generated. Many methods of makingantibodies are known to persons of skill. The following discussion ispresented as a general overview of the techniques available; however,one of skill will recognize that many variations upon the followingmethods are known.

1. Antibody Production

A number of immunogens are used to produce antibodies specificallyreactive with mesothelin polypeptides. Recombinant or syntheticpolypeptides of 10 amino length, or greater, selected from sub-sequencesof SEQ ID NO: 1 are the preferred polypeptide immunogen for theproduction of monoclonal or polyclonal antibodies. In one class ofpreferred embodiments, an immunogenic peptide conjugate is also includedas an immunogen. Naturally occurring polypeptides are also used eitherin pure or impure form. Transfected mammalian cells overexpressingrecombinant mesothelin can also be used as an immunogen, either in wholeintact cells or membrane preparations. These immunogens are useful forpolyclonal or monoclonal antibody generation.

Recombinant polypeptides are expressed in eukaryotic or prokaryoticcells and purified using standard techniques. The polypeptide, or asynthetic version thereof, is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies can begenerated for subsequent use in immunoassays to measure the presence andquantity of the polypeptide.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen, preferably a purified polypeptide, apolypeptide coupled to an appropriate carrier (e.g., GST, keyhole limpethemanocyanin, etc.), or a polypeptide incorporated into an immunizationvector such as a recombinant vaccinia virus (see, U.S. Pat. No.4,722,848) is mixed with an adjuvant and animals are immunized with themixture. The animals immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the polypeptide of interest. When appropriately high titers ofantibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the polypeptide is performed wheredesired. See, e.g., Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A LaboratoryManual Cold Spring Harbor Press, NY, which are incorporated herein byreference, and the examples below.

Antibodies, including binding fragments and single chain recombinantversions thereof, against predetermined fragments of mesothelinpolypeptides are raised by immunizing animals, e.g., with conjugates ofthe fragments with carrier proteins as described above. Typically, theimmunogen of interest is a peptide of at least about 3 amino acids, moretypically the peptide is 5 amino acids in length, preferably, thefragment is 10 amino acids in length and more preferably the fragment is15 amino acids in length or greater. The peptides are typically coupledto a carrier protein (e.g., as a fusion protein), or are recombinantlyexpressed in an immunization or expression vector. Antigenicdeterminants on peptides to which antibodies bind are typically 3 to 10amino acids in length.

Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies are screened for binding to normal ormodified polypeptides. Specific monoclonal and polyclonal antibodieswill usually bind with a K_(D) of at least about 0.1 mM, more usually atleast about 50 μM, and most preferably at least about 1 μM or better.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arewell known and are found in, e.g., Asai, ed. Antibodies in Cell Biology,Academic Press, Inc., San Diego, Calif.; Stites et al. (eds.) Basic andClinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, Supra; Goding(1986) Monoclonal Antibodies: Principles and Practice (2d ed.) AcademicPress, New York, N.Y.; and Kohler and Milstein (1975) Nature 256:495-497. The polypeptides and antibodies of the present invention areused with or without modification, and include chimeric antibodies suchas humanized murine antibodies.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al. (1989) Science246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546.

Frequently, the polypeptides and antibodies will be labeled by joining,either covalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionucleotides, enzymes,substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, magnetic particles, and the like. Patentsteaching the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.Also, recombinant immunoglobulins may be produced. See, for example,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'lAcad. Sci. USA 86: 10029-10033.

The antibodies of this invention are also used for affinitychromatography in isolating mesothelin polypeptides. Columns areprepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified mesothelinpolypeptides are released.

The antibodies can be used to screen expression libraries for particularexpression products such as mammalian mesothelin. Usually the antibodiesin such a procedure are labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies raised against mesothelin polypeptides can also be used toraise anti-idiotypic antibodies. These are useful for detecting ordiagnosing various pathological conditions related to the presence ofthe respective antigens.

2. Immunoassays

A particular protein can be quantified by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures ingeneral, see Stites and Terr (eds.) 1991 Basic and Clinical Immunology(7th ed.). Moreover, the immunoassays of the present invention can beperformed in any of several configurations, e.g., those reviewed inMaggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.;Tijan (1985) “Practice and Theory of Enzyme Immunoassays,” LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers B.V., Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987)Immunoassay: A Practical Guide Academic Press, Orlando, Fla.; Price andNewman (eds.) (1991) Principles and Practice of Immunoassays StocktonPress, NY; and Ngo (ed.) (1988) Non-isotopic Immunoassays Plenum Press,NY.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledmesothelin peptide or a labeled anti-mesothelin antibody. Alternatively,the labeling agent may be a third moiety, such as another antibody, thatspecifically binds to the antibody/mesothelin complex, or to a modifiedcapture group (e.g., biotin) which is covalently linked to themesothelin peptide or anti-mesothelin antibody.

In a preferred embodiment, the labeling agent is an antibody thatspecifically binds to the capture agent (anti-mesothelin). Such agentsare well known to those of skill in the art, and most typically compriselabeled antibodies that specifically bind antibodies of the particularanimal species from which the capture agent is derived (e.g., ananti-idiotypic antibody). Thus, for example, where the capture agent isa mouse derived anti-human mesothelin antibody, the label agent may be agoat anti-mouse IgG, i.e., an antibody specific to the constant regionof the mouse antibody.

Other proteins capable of specifically binding immunoglobulin constantregions, such as streptococcal protein A or protein G are also used asthe labeling agent. These proteins are normal constituents of the cellwalls of streptococcal bacteria. They exhibit a strong non-immunogenicreactivity with immunoglobulin constant regions from a variety ofspecies. See, generally Kronval, et al., (1973) J. Immunol.,111:1401-1406, and Akerstrom, et al., (1985) J. Immunol., 135:2589-2542.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays are carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 5° C. to 45° C.

(a) Non-Competitive Assay Formats

Immunoassays for detecting mesothelin may be either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured analyte (in this case mesothelin) is directlymeasured. In one preferred “sandwich” assay, for example, the captureagent (e.g., anti-mesothelin antibodies) are bound directly to a solidsubstrate where they are immobilized. These immobilized antibodies thencapture mesothelin present in the test sample. The mesothelin thusimmobilized is then bound by a labeling agent, such as a second humanmesothelin antibody bearing a label. Alternatively, the secondmesothelin antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived.

Sandwich assays for mesothelin may be constructed. As described above,the immobilized anti-mesothelin specifically binds to mesothelin presentin the sample. The labeled anti-mesothelin then binds to the alreadybound mesothelin. Free labeled anti-mesothelin is washed away and theremaining bound labeled anti-mesothelin is detected (e.g., using a gammadetector where the label is radioactive).

(b) Competitive Assay Formats

In competitive assays, the amount of analyte (e.g., mesothelin) presentin the sample is measured indirectly by measuring the amount of an added(exogenous) analyte displaced (or competed away) from a capture agent(e.g., anti-mesothelin antibody) by the analyte present in the sample.In one competitive assay, a known amount of analyte is added to thesample and the sample is contacted with a capture agent, in this case anantibody that specifically binds the analyte. The amount of analytebound to the antibody is inversely proportional to the concentration ofanalyte present in the sample.

In a particularly preferred embodiment, the capture agent is immobilizedon a solid substrate. The amount of mesothelin bound to the captureagent is determined either by measuring the amount of mesothelin presentin an mesothelin/antibody complex, or alternatively by measuring theamount of remaining uncomplexed mesothelin. The amount of mesothelin maybe detected by providing a labeled mesothelin.

A hapten inhibition assay is another preferred competitive assay. Inthis assay, a known analyte, in this case mesothelin, is immobilized ona solid substrate. A known amount of anti-mesothelin antibody is addedto the sample, and the sample is then contacted with the immobilizedmesothelin. In this case, the amount of anti-mesothelin antibody boundto the immobilized mesothelin is proportional to the amount ofmesothelin present in the sample. Again the amount of immobilizedantibody is detected by detecting either the immobilized fraction ofantibody or the fraction of the antibody that remains in solution.Detection may be direct where the antibody is labeled, or indirect bythe subsequent addition of a labeled moiety that specifically binds tothe antibody as described above.

(c) Generation of Pooled Antisera for Use in Immunoassays.

A mesothelin protein that specifically binds to or that is specificallyimmunoreactive with an antibody generated against a defined immunogen,such as an immunogen consisting of the amino acid sequence of SEQ ID NO:2, is determined in an immunoassay. The immunoassay uses a polyclonalantiserum which was raised to the protein of SEQ ID NO: 2 (theimmunogenic polypeptide).

In order to produce an antisera for use in an immunoassay, thepolypeptide of SEQ ID NO: 2 is isolated as described herein. Forexample, recombinant protein can be produced in a mammalian or othereukaryotic cell line. An inbred strain of mice is immunized with theprotein of SEQ ID NO: 2 using a standard adjuvant, such as Freund'sadjuvant, and a standard mouse immunization protocol (see Harlow andLane, supra). Alternatively, a synthetic polypeptide derived from thesequences disclosed herein and conjugated to a carrier protein is usedas an immunogen. Polyclonal sera are collected and titered against theimmunogenic polypeptide in an immunoassay, for example, a solid phaseimmunoassay with the immunogen immobilized on a solid support.Polyclonal antisera with a titer of 10⁴ or greater are selected andtested for their cross reactivity against proteins of interest, using acompetitive binding immunoassay such as the one described in Harlow andLane, supra, at pages 570-573.

Immunoassays in the competitive binding format are used forcrossreactivity determinations. For example, the immunogenic polypeptideis immobilized to a solid support. Proteins added to the assay competewith the binding of the antisera to the immobilized antigen. The abilityof the above proteins to compete with the binding of the antisera to theimmobilized protein is compared to the immunogenic polypeptide. Thepercent crossreactivity for the above proteins is calculated, usingstandard calculations. Those antisera with less than 10% crossreactivitywith the protein of interest are combined and pooled. The cross-reactingantibodies are then removed from the pooled antisera byimmunoadsorbtion. The immunoadsorbed and pooled antisera are then usedin a competitive binding immunoassay as described herein to compare asecond “target” polypeptide to the immunogenic polypeptide. In order tomake this comparison, the two polypeptides are each assayed at a widerange of concentrations and the amount of each polypeptide required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined using standard techniques. If the amount of the targetpolypeptide required is less than twice the amount of the immunogenicpolypeptide that is required, then the target polypeptide is said tospecifically bind to an antibody generated to the immunogenic protein.As a final determination of specificity, the pooled antisera is fullyimmunoadsorbed with the immunogenic polypeptide until no binding to thepolypeptide used in the immunoadsorbtion is detectable. The fullyimmunoadsorbed antisera is then tested for reactivity with the testpolypeptide. If no reactivity is observed, then the test polypeptide isspecifically bound by the antisera elicited by the immunogenic protein.

D. Other Assay Formats

Western blot analysis can also be used to detect and quantify thepresence of mesothelin in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind mesothelin. The anti-mesothelin antibodiesspecifically bind to mesothelin on the solid support. These antibodiesmay be directly labeled or alternatively may be subsequently detectedusing labeled antibodies (e.g., labeled sheep anti-mouse antibodieswhere the antibody to mesothelin is a murine antibody) that specificallybind to the anti-mesothelin.

Other assay formats include liposome immunoassays (LIAs), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,(1986) Amer. Clin. Prod. Rev. 5:34-41), which is incorporated herein byreference.

E. Labels

The labeling agent for the applications described herein can be, e.g., amonoclonal antibody, a polyclonal antibody, a mesothelin binding proteinor complex such as those described herein, or a polymer such as anaffinity matrix, carbohydrate or lipid. Detection may proceed by anyknown method, such as immunoblotting, western analysis, gel-mobilityshift assays, fluorescent in situ hybridization analysis (FISH),tracking of radioactive or bioluminescent markers, nuclear magneticresonance, electron paramagnetic resonance, stopped-flow spectroscopy,column chromatography, capillary electrophoresis, or other methods whichtrack a molecule based upon an alteration in size and/or charge. Theparticular label or detectable group used in the assay is not a criticalaspect of the invention. The detectable group can be any material havinga detectable physical or chemical property. Such detectable labels havebeen well-developed in the field of immunoassays and, in general, anylabel useful in such methods can be applied to the present invention.Thus, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, texas red, rhodamine, and the like), radiolabels (e.g.,³H, ¹²⁵, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase,alkaline phosphatase and others commonly used in an ELISA), andcalorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, ease of conjugation of thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabelling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence, e.g., by microscopy,visual inspection, via photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providingappropriate substrates for the enzyme and detecting the resultingreaction product. Finally, simple colorimetric labels may be detectedsimply by observing the color associated with the label. Thus, invarious dipstick assays, conjugated gold often appears pink, whilevarious conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

F. Substrates

As mentioned above, depending upon the assay, various components,including the antigen, target antibody, or anti-human antibody, may bebound to a solid surface. Many methods for immobilizing biomolecules toa variety of solid surfaces are known in the art. For instance, thesolid surface may be a membrane (e.g., nitrocellulose), a microtiterdish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass orplastic), a dipstick (e.g. glass, PVC, polypropylene, polystyrene,latex, and the like), a microcentrifuge tube, or a glass, silica,plastic, metallic or polymer bead. The desired component may becovalently bound, or noncovalently attached through nonspecific bonding.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

In preparing the surface, a plurality of different materials may beemployed, e.g., as laminates, to obtain various properties. For example,protein coatings, such as gelatin can be used to avoid non-specificbinding, simplify covalent conjugation, enhance signal detection or thelike.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature. See, for example, Immobilized Enzymes, Ichiro Chibata,Halsted Press, New York, 1978, and Cuatrecasas, J. Biol. Chem. 245 3059(1970) which are incorporated herein by reference.

In addition to covalent bonding, various methods for noncovalentlybinding an assay component can be used. Noncovalent binding is typicallynonspecific absorption of a compound to the surface. Typically, thesurface is blocked with a second compound to prevent nonspecific bindingof labeled assay components. Alternatively, the surface is designed suchthat it nonspecifically binds one component but does not significantlybind another. For example, a surface bearing a lectin such asConcanavalin A will bind a carbohydrate containing compound but not alabeled protein that lacks glycosylation. Various solid surfaces for usein noncovalent attachment of assay components are reviewed in U.S. Pat.Nos. 4,447,576 and 4,254,082, which are incorporated herein byreference.

II. Targeting Effector Molecules to Mesothelin

This invention also provides for compositions and methods for detectingthe presence or absence of tumor cells bearing mesothelin. These methodsinvolve providing a chimeric molecule comprising an effector molecule,that is a detectable label attached to a targeting molecule thatspecifically binds mesothelin. The mesothelin targeting moietyspecifically binds the chimeric molecule to tumor cells which are thenmarked by their association with the detectable label. Subsequentdetection of the cell-associated label indicates the presence of a tumorcell.

In yet another embodiment, the effector molecule may be another specificbinding moiety such as an antibody, a growth factor, or a ligand. Thechimeric molecule will then act as a highly specific bifunctionallinker. This linker may act to bind and enhance the interaction betweencells or cellular components to which the fusion protein binds. Thus,for example, where the “targeting” component of the chimeric moleculecomprises a polypeptide that specifically binds to mesothelin and the“effector” component is an antibody or antibody fragment (e.g. an Fvfragment of an antibody), the targeting component specifically bindscancer cells, while the effector component inhibits cell growth or mayact to enhance and direct an immune response toward target cancer cells.

In still yet another embodiment the effector molecule may be apharmacological agent (e.g. a drug) or a vehicle containing apharmacological agent. Thus, the moiety that specifically binds tomesothelin may be conjugated to a drug such as vinblastine, doxirubicin,genistein (a tyrosine kinase inhibitor), an antisense molecule, andother pharmacological agents known to those of skill in the art, therebyspecifically targeting the pharmacological agent to tumor cells.

Alternatively, the targeting molecule may be bound to a vehiclecontaining the therapeutic composition. Such vehicles include, but arenot limited to liposomes, micelles, various synthetic beads, and thelike.

One of skill in the art will appreciate that the chimeric molecules ofthe present invention may include multiple targeting moieties bound to asingle effector or conversely, multiple effector molecules bound to asingle targeting moiety. In still other embodiments, the chimericmolecules may include both multiple targeting moieties and multipleeffector molecules. Thus, for example, this invention provides for “dualtargeted” cytotoxic chimeric molecules in which targeting molecule thatspecifically binds to mesothelin is attached to a cytotoxic molecule andanother molecule (e.g. an antibody, or another ligand) is attached tothe other terminus of the toxin. Such a dual-targeted cytotoxin mightcomprise a growth factor substituted for domain Ia, for example, at theamino terminus of a PE and anti-TAC(Fv) inserted in domain III, betweenamino acid 604 and 609. Other antibodies may also be suitable.

A. The Targeting Molecule

In a preferred embodiment, the targeting molecule is a molecule thatspecifically binds to mesothelin. A variety of immunoassay formats maybe used to select appropriate antibodies and are discussed above.

B. The Effector Molecule

As described above, the effector molecule component of the chimericmolecules of this invention may be any molecule whose activity it isdesired to deliver to cells that express mesothelin. Particularlypreferred effector molecules include cytotoxins such as PE or DT,radionuclides, ligands such as growth factors, antibodies, detectablelabels such as fluorescent or radioactive labels, and therapeuticcompositions such as liposomes and various drugs.

1. Cytotoxins

Particularly preferred cytotoxins include Pseudomonas exotoxins,Diphtheria toxins, ricin, and abrin. Pseudomonas exotoxin and Diphtheriatoxin are most preferred.

(a) Pseudomonas Exotoxin (PE)

Pseudomonas exotoxin A (PE) is an extremely active monomeric protein(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, whichinhibits protein synthesis in eukaryotic cells through the inactivationof elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation(catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD ontoEF-2).

The toxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2, which inactivates the protein andcauses cell death. The function of domain Ib (amino acids 365-399)remains undefined, although a large part of it, amino acids 365-380, canbe deleted without loss of cytotoxicity. See Siegall et al., J. Biol.Chem. 264: 14256-14261 (1989), incorporated by reference herein.

Where the targeting molecule is fused to PE, a preferred PE molecule isone in which domain Ia (amino acids 1 through 252) is deleted and aminoacids 365 to 380 have been deleted from domain Ib. However all of domainIb and a portion of domain II (amino acids 350 to 394) can be deleted,particularly if the deleted sequences are replaced with a linkingpeptide such as GGGGS (SEQ ID NO: 3).

In addition, the PE molecules can be further modified usingsite-directed mutagenesis or other techniques known in the art, to alterthe molecule for a particular desired application. Means to alter the PEmolecule in a manner that does not substantially affect the functionaladvantages provided by the PE molecules described here can also be usedand such resulting molecules are intended to be covered herein.

For maximum cytotoxic properties of a preferred PE molecule, severalmodifications to the molecule are recommended. An appropriate carboxylterminal sequence to the recombinant molecule is preferred totranslocate the molecule into the cytosol of target cells. Amino acidsequences which have been found to be effective include REDLK (SEQ IDNO:4) (as in native PE), REDL (SEQ ID NO: 5), RDEL (SEQ ID NO: 6), orKDEL (SEQ ID NO: 7), repeats of those, or other sequences that functionto maintain or recycle proteins into the endoplasmic reticulum, referredto here as “endoplasmic retention sequences”. See, for example,Chaudhary et al, Proc. Natl. Acad. Sci. USA 87:308-312 and Seetharam etal, J. Biol. Chem. 266: 17376-17381 (1991) and commonly assigned, U.S.Ser. No. 07/459,635 filed Jan. 2, 1990, all of which are incorporated byreference herein.

Deletions of amino acids 365-380 of domain Ib can be made without lossof activity. Further, amino acids 1-279 may be deleted so that the toxinbegins with a methionine followed by glycine at position 280. A serinemay be placed at position 289 to prevent formation of improper disulfidebonds is beneficial. The targeting molecule may be inserted inreplacement for domain Ia.

Preferred forms of PE contain amino acids 253-364 and 381/608, and arefollowed by the native sequences REDLK (SEQ ID NO: 4) or the mutantsequences KDEL (SEQ ID NO: 7) or RDEL (SEQ ID NO: 6). Lysines atpositions 590 and 606 may or may not be mutated to glutamine.

The targeting molecule may also be inserted at a point within domain IIIof the PE molecule. Most preferably the targeting molecule is fusedbetween about amino acid positions 607 and 609 of the PE molecule. Thismeans that the targeting molecule is inserted after about amino acid 607of the molecule and an appropriate carboxyl end of PE is recreated byplacing amino acids about 604-613 of PE after the targeting molecule.Thus, the targeting molecule is inserted within the recombinant PEmolecule after about amino acid 607 and is followed by amino acids604-613 of domain III. The targeting molecule may also be inserted intodomain Ib to replace sequences not necessary for toxicity. Debinski, etal. Mol. Cell. Biol., 11: 1751-1753 (1991).

Methods of cloning genes encoding PE fused to various ligands are wellknown to those of skill in the art. See, for example, Siegall et al.,FASEB J., 3: 2647-2652 (1989); Chaudhary et al. Proc. Natl. Acad. Sci.USA, 84: 4538-4542 (1987), which are incorporated herein by reference.

Those skilled in the art will realize that additional modifications,deletions, insertions and the like may be made to the chimeric moleculesof the present invention or to the nucleic acid sequences encodingmesothelin-directed chimeric molecules. All such constructions may bemade by methods of genetic engineering well known to those skilled inthe art (see, generally, Sambrook et al., supra) and may produceproteins that have differing properties of affinity, specificity,stability and toxicity that make them particularly suitable for variousclinical or biological applications.

(b) Diphtheria Toxin (DT)

Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 thereby inhibiting protein synthesis. Diphtheriatoxin, however, is divided into two chains, A and B, linked by adisulfide bridge. In contrast to PE, chain B of DT, which is on thecarboxyl end, is responsible for receptor binding and chain A, which ispresent on the amino end, contains the enzymatic activity (Uchida etal., Science, 175: 901-903 (1972); Uchida et al. J. Biol. Chem., 248:3838-3844 (1973)).

In a preferred embodiment, the targeting molecule-Diphtheria toxinfusion proteins of this invention have the native receptor-bindingdomain removed by truncation of the Diphtheria toxin B chain.Particularly preferred is DT388, a DT in which the carboxyl terminalsequence beginning at residue 389 is removed. Chaudhary, et al., Bioch.Biophys. Res. Comm., 180: 545-551 (1991).

Like the PE chimeric cytotoxins, the DT molecules may be chemicallyconjugated to a mesothelin targeting molecule, but, in a preferredembodiment, the targeting molecule will be fused to the Diphtheria toxinby recombinant means. The genes encoding protein chains may be cloned incDNA or in genomic form by any cloning procedure known to those skilledin the art. Methods of cloning genes encoding DT fused to variousligands are also well known to those of skill in the art. See, forexample, Williams et al. J. Biol. Chem. 265: 11885-11889 (1990) andcopending patent application (U.S. Ser. No. 07/620,939) which describethe expression of a number of growth-factor-DT fusion proteins.

The term “Diphtheria toxin” (DT) as used herein refers to full lengthnative DT or to a DT that has been modified. Modifications typicallyinclude removal of the targeting domain in the B chain and, morespecifically, involve truncations of the carboxyl region of the B chain.

Detectable labels suitable for use as the effector molecule component ofthe chimeric molecules of this invention include any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means all as described above.

C. Attachment of the Targeting Molecule to the Effector Molecule

One of skill will appreciate that the targeting molecule and effectormolecules may be joined together in any order. Thus, where the targetingmolecule is a polypeptide, the effector molecule may be joined to eitherthe amino or carboxy termini of the targeting molecule. The targetingmolecule may also be joined to an internal region of the effectormolecule, or conversely, the effector molecule may be joined to aninternal location of the targeting molecule, as long as the attachmentdoes not interfere with the respective activities of the molecules.

The targeting molecule and the effector molecule may be attached by anyof a number of means well known to those of skill in the art. Typicallythe effector molecule is conjugated, either directly or through a linker(spacer), to the targeting molecule. However, where both the effectormolecule and the targeting molecule are polypeptides it is preferable torecombinantly express the chimeric molecule as a single-chain fusionprotein.

D. Conjugation of the Effector Molecule to the Targeting Molecule

In one embodiment, the targeting molecule is chemically conjugated tothe effector molecule (e.g. a cytotoxin, a label, a ligand, or a drug orliposome). Means of chemically conjugating molecules are well known tothose of skill.

The procedure for attaching an agent to an antibody or other polypeptidetargeting molecule will vary according to the chemical structure of theagent. Polypeptides typically contain variety of functional groups;e.g., carboxylic acid (COOH) or free amine (—NH₂) groups, which areavailable for reaction with a suitable functional group on an effectormolecule to bind the effector thereto.

Alternatively, the targeting molecule and/or effector molecule may bederivatized to expose or attach additional reactive functional groups.The derivatization may involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, is a molecule that is used to join thetargeting molecule to the effector molecule. The linker is capable offorming covalent bonds to both the targeting molecule and to theeffector molecule. Suitable linkers are well known to those of skill inthe art and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Wherethe targeting molecule and the effector molecule are polypeptides, thelinkers may be joined to the constituent amino acids through their sidegroups (e.g., through a disulfide linkage to cysteine). However, in apreferred embodiment, the linkers will be joined to the alpha carbonamino and carboxyl groups of the terminal amino acids.

A bifunctional linker having one functional group reactive with a groupon a particular agent, and another group reactive with an antibody, maybe used to form the desired immunoconjugate. Alternatively,derivatization may involve chemical treatment of the targeting molecule,e.g., glycol cleavage of the sugar moiety of a the glycoprotein antibodywith periodate to generate free aldehyde groups. The free aldehydegroups on the antibody may be reacted with free amine or hydrazinegroups on an agent to bind the agent thereto. (See U.S. Pat. No.4,671,958). Procedures for generation of free sulfhydryl groups onpolypeptide, such as antibodies or antibody fragments, are also known(See U.S. Pat. No. 4,659,839).

Many procedures and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins suchas antibodies are known. See, for example, European Patent ApplicationNo. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. Cancer Res.47: 4071-4075 (1987) which are incorporated herein by reference. Inparticular, production of various immunotoxins is well-known within theart and can be found, for example in “Monoclonal Antibody-ToxinConjugates: Aiming the Magic Bullet,” Thorpe et al., MonoclonalAntibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982),Waldmann, Science, 252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and4,894,443 which are incorporated herein by reference.

In some circumstances, it is desirable to free the effector moleculefrom the targeting molecule when the chimeric molecule has reached itstarget site. Therefore, chimeric conjugates comprising linkages whichare cleavable in the vicinity of the target site may be used when theeffector is to be released at the target site. Cleaving of the linkageto release the agent from the antibody may be prompted by enzymaticactivity or conditions to which the immunoconjugate is subjected eitherinside the target cell or in the vicinity of the target site. When thetarget site is a tumor, a linker which is cleavable under conditionspresent at the tumor site (e.g. when exposed to tumor-associated enzymesor acidic pH) may be used.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers which are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient's complementsystem. In view of the large number of methods that have been reportedfor attaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

E. Production of Fusion Proteins

Where the targeting molecule and/or the effector molecule is relativelyshort (i.e., less than about 50 amino acids) they may be synthesizedusing standard chemical peptide synthesis techniques. Where bothmolecules are relatively short the chimeric molecule may be synthesizedas a single contiguous polypeptide. Alternatively the targeting moleculeand the effector molecule may be synthesized separately and then fusedby condensation of the amino terminus of one molecule with the carboxylterminus of the other molecule thereby forming a peptide bond.Alternatively, the targeting and effector molecules may each becondensed with one end of a peptide spacer molecule thereby forming acontiguous fusion protein.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984) which are incorporated herein by reference.

In a preferred embodiment, the chimeric fusion proteins of the presentinvention are synthesized using recombinant DNA methodology. Generallythis involves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion proteins of this invention may be prepared byany suitable method, including, for example, cloning and restriction ofappropriate sequences or direct chemical synthesis by methods such asthe phosphotriester method of Narang et al. Meth. Enzymol. 68: 90-99(1979); the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage et al.,Tetra. Lett., 22: 1859-1862 (1981); and the solid support method of U.S.Pat. No. 4,458,066, all incorporated by reference herein.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

While the two molecules are preferably essentially directly joinedtogether, one of skill will appreciate that the molecules may beseparated by a peptide spacer consisting of one or more amino acids.Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

The nucleic acid sequences encoding the fusion proteins may be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene will beoperably linked to appropriate expression control sequences for eachhost. For E. coli this includes a promoter such as the T7, trp, orlambda promoters, a ribosome binding site and preferably a transcriptiontermination signal. For eukaryotic cells, the control sequences willinclude a promoter and preferably an enhancer derived fromimmunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylationsequence, and may include splice donor and acceptor sequences.

The plasmids and vectors of the invention can be transferred into thechosen host cell by well-known methods such as calcium chloridetransformation for E. coli and calcium phosphate treatment orelectroporation for mammalian cells. Cells transformed by the plasmidscan be selected by resistance to antibiotics conferred by genescontained on the plasmids, such as the amp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

III. Administration to Patients of Targeting Agents to Mesothelin

Therapeutic agents of the present invention, such as antibodies tomesothelin or such as antibodies or other targeting molecules attachedto an effector molecule are administered in any suitable manner,preferably with pharmaceutically acceptable carriers. One skilled in theart will appreciate that suitable methods of administering suchcompounds in the context of the present invention to a patient areavailable, and, although more than one route can be used to administer aparticular compound, a particular route can often provide a moreimmediate and more effective reaction than another route. It should berecognized that the administration of peptides are well-known for avariety of diseases, and one of skill is able to extrapolate theinformation available for use of peptides to treat these other diseasesto mesothelin peptides.

Pharmaceutically acceptable carriers are also well known to those whoare skilled in the art. The optimal choice of carrier will be determinedin part by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there is a wide varietyof suitable formulations of the pharmaceutical compositions of thepresent invention.

Antibodies may be formulated into an injectable preparation. Parenteralformulations are known and are suitable for use in the invention,preferably for i.m. or i.v. administration. The formulations containingtherapeutically effective amounts of antibodies or immunotoxins areeither sterile liquid solutions, liquid suspensions or lyophilizedversions and optionally contain stabilizers or excipients. Lyophilizedcompositions are reconstituted with suitable diluents, e.g., water forinjection, saline, 0.3% glycine and the like, at a level of about from0.01 mg/kg of host body weight to 10 mg/kg where appropriate. Typically,the pharmaceutical compositions containing the antibodies orimmunotoxins will be administered in a therapeutically effective dose ina range of from about 0.01 mg/kg to about 5 mg/kg of the treated mammal.A preferred therapeutically effective dose of the pharmaceuticalcomposition containing antibody or immunotoxin will be in a range offrom about 0.01 mg/kg to about 0.5 mg/kg body weight of the treatedmammal administered over several days to two weeks by daily intravenousinfusion, each given over a one hour period, in a sequential patientdose-escalation regimen.

Antibody may be administered systemically by injection i.m.,subcutaneously, intrathecally or intraperitoneally or into vascularspaces, particularly into the peritoneal cavity or thoracic cavity,e.g., injection at a dosage of greater than about 1 μg/cc fluid/day. Apermanent intrathecal catheter would be a convenient means to administertherapeutic antibodies. The dose will be dependent upon the propertiesof the antibody or immunotoxin employed, e.g., its activity andbiological half-life, the concentration of antibody in the formulation,the site and rate of dosage, the clinical tolerance of the patientinvolved, the disease afflicting the patient and the like as is wellwithin the skill of the physician.

The antibody of the present invention may be administered in solution.The pH of the solution should be in the range of pH 5 to 9.5, preferablypH 6.5 to 7.5. The antibody or derivatives thereof should be in asolution having a suitable pharmaceutically acceptable buffer such asphosphate, tris (hydroxymethyl) aminomethane-HCl or citrate and thelike. Buffer concentrations should be in the range of 1 to 100 mM. Thesolution of antibody may also contain a salt, such as sodium chloride orpotassium chloride in a concentration of 50 to 150 mM. An effectiveamount of a stabilizing agent such as an albumin, a globulin, a gelatin,a protamine or a salt of protamine may also be included and may be addedto a solution containing antibody or immunotoxin or to the compositionfrom which the solution is prepared. Antibody or immunotoxin may also beadministered via microspheres, liposomes or other microparticulatedelivery systems placed in certain tissues including blood.

Dosages

In therapeutic applications, the dosages of compounds used in accordancewith the invention vary depending on the class of compound and thecondition being treated. The age, weight, and clinical condition of therecipient patient; and the experience and judgment of the clinician orpractitioner administering the therapy are among the factors affectingthe selected dosage. For example, the dosage of an immunoglobulin canrange from about 0.1 milligram per kilogram of body weight per day toabout 10 mg/kg per day for polyclonal antibodies and about 5% to about20% of that amount for monoclonal antibodies. In such a case, theimmunoglobulin can be administered once daily as an intravenousinfusion. Preferably, the dosage is repeated daily until either atherapeutic result is achieved or until side effects warrantdiscontinuation of therapy. Generally, the dose should be sufficient totreat or ameliorate symptoms or signs of the disease without producingunacceptable toxicity to the patient.

A therapeutically effective amount of the compound is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement, such as inhibition of tumor-cell growth, asnoted by the clinician or other qualified observer. The dosing rangevaries with the compound used, the route of administration and thepotency of the particular compound.

IV. Gene Therapy and Inhibitory Nucleic Acid Therapeutics

Using the nucleotide sequence information of this invention, one skilledin the art can formulate strategies and methods to isolate themesothelin gene, describe the gene structure for function, and may alsodiscover specific promoters for known or unknown transcriptional factorswhich may be of further value in the genetic intervention ofmesothelioma and ovarian cancers. Analytical DNA sequencing of normalmesothelin in mesothelial cells may lead to a discovery of mutation(s)of the gene in mesothelioma and ovarian cancers.

Mesothelin manifests an adhesive property which can be attributed toimplantation of the mesothelioma and ovarian cancers. By introducingantisense DNA or blocking the transcription of mesothelin gene, novelgene therapy regimens can be set up according to current strategies ofgene therapy.

Inhibitory nucleic acid therapeutics which can block the expression oractivity of the mesothelin gene will be useful in slowing or inhibitingthe growth of mesotheliomas or ovarian tumors or other abnormal cellswhich are associated with mesothelin. Inhibitory nucleic acids may besingle-stranded nucleic acids, which can specifically bind to acomplementary nucleic acid sequence. By binding to the appropriatetarget sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex or triplex isformed. These nucleic acids are often termed “antisense” because theyare usually complementary to the sense or coding strand of the gene,although recently approaches for use of “sense” nucleic acids have alsobeen developed. The term “inhibitory nucleic acids” as used herein,refers to both “sense” and “antisense” nucleic acids.

By binding to the target nucleic acid, the inhibitory nucleic acid caninhibit the function of the target nucleic acid. This could, forexample, be a result of blocking DNA transcription, processing orpoly(A) addition to mRNA, DNA replication, translation, or promotinginhibitory mechanisms of the cells, such as promoting RNA degradation.Inhibitory nucleic acid methods therefore encompass a number ofdifferent approaches to altering expression of, for example, amesothelin gene. These different types of inhibitory nucleic acidtechnology are described in Helene, C. and Toulme, J., 1990, Biochim.Biophys. Acta. 1049:99-125, which is hereby incorporated by referenceand is referred to hereinafter as “Helene and Toulme.”

In brief, inhibitory nucleic acid therapy approaches can be classifiedinto those that target DNA sequences, those that target RNA sequences(including pre-mRNA and mRNA), those that target proteins (sense strandapproaches), and those that cause cleavage or chemical modification ofthe target nucleic acids.

Approaches targeting DNA fall into several categories. Nucleic acids canbe designed to bind to the duplex DNA to form a triple helical or“triplex” structure. Alternatively, inhibitory nucleic acids aredesigned to bind to regions of single stranded DNA resulting from theopening of the duplex DNA during replication or transcription. SeeHelene and Toulme.

More commonly, inhibitory nucleic acids are designed to bind to mRNA ormRNA precursors. Inhibitory nucleic acids are used to prevent maturationof pre-mRNA. Inhibitory nucleic acids may be designed to interfere withRNA processing, splicing or translation.

The inhibitory nucleic acids can be targeted to mRNA. In this approach,the inhibitory nucleic acids are designed to specifically blocktranslation of the encoded protein. Using this approach, the inhibitorynucleic acid can be used to selectively suppress certain cellularfunctions by inhibition of translation of mRNA encoding criticalproteins. For example, an inhibitory nucleic acid complementary toregions of c-myc mRNA inhibits c-myc protein expression in a humanpromyelocytic leukemia cell line, HL60, which overexpresses the c-mycproto-oncogene. See Wickstrom E. L., et al., 1988, PNAS (USA)85:1028-1032 and Harel-Bellan, A., et al., 1988, Exp. Med.168:2309-2318. As described in Helene and Toulme, inhibitory nucleicacids targeting mRNA have been shown to work by several differentmechanisms to inhibit translation of the encoded protein(s).

The inhibitory nucleic acids introduced into the cell can also encompassthe “sense” strand of the gene or mRNA to trap or compete for theenzymes or binding proteins involved in mRNA translation. See Helene andToulme.

Lastly, the inhibitory nucleic acids can be used to induce chemicalinactivation or cleavage of the target genes or mRNA. Chemicalinactivation can occur by the induction of crosslinks between theinhibitory nucleic acid and the target nucleic acid within the cell.Other chemical modifications of the target nucleic acids induced byappropriately derivatized inhibitory nucleic acids may also be used.

Cleavage, and therefore inactivation, of the target nucleic acids may beaccomplished by attaching a substituent to the inhibitory nucleic acidwhich can be activated to induce cleavage reactions. The substituent canbe one that affects either chemical, or enzymatic cleavage.Alternatively, cleavage can be induced by the use of ribozymes orcatalytic RNA. In this approach, the inhibitory nucleic acids wouldcomprise either naturally occurring RNA (ribozymes) or synthetic nucleicacids with catalytic activity.

The targeting of inhibitory nucleic acids to specific cells of theimmune system by conjugation with targeting moieties binding receptorson the surface of these cells can be used for all of the above forms ofinhibitory nucleic acid therapy. This invention encompasses all of theforms of inhibitory nucleic acid therapy as described above and asdescribed in Helene and Toulme.

This invention relates to the targeting of inhibitory nucleic acids tosequences of mesothelin for use in inhibiting or slowing the growth oftumors associated with mesothelin. A problem associated with inhibitorynucleic acid therapy is the effective delivery of the inhibitory nucleicacid to the target cell in vivo and the subsequent internalization ofthe inhibitory nucleic acid by that cell. Delivery, however, can beaccomplished by linking the inhibitory nucleic acid to a targetingmoiety to form a conjugate that binds to a specific receptor on thesurface of the target infected cell, and which is internalized afterbinding. Preferably, the inhibitory nucleic acid will be delivered tothe peritoneal cavity, the thoracic cavity, as well as any otherlocation where cells bearing mesothelin are of interest.

Gene therapy can also correct genetic defects by insertion of exogenouscellular genes that encode a desired function into cells that lack thatfunction, such that the expression of the exogenous gene a) corrects agenetic defect or b) causes the destruction of cells that aregenetically defective. Methods of gene therapy are well known in theart, see, for example, Lu, M., et al. (1994), Human Gene Therapy 5:203;Smith, C. (1992), J. Hematotherapy 1:155; Cassel, A., et al. (1993),Exp. Hematol. 21-:585 (1993); Larrick, J. W. and Burck, K. L., GENETHERAPY: APPLICATION OF MOLECULAR BIOLOGY, Elsevier Science PublishingCo., Inc., New York, N.Y. (1991) and Kreigler, M. GENE TRANSFER ANDEXPRESSION: A LABORATORY MANUAL, W.H. Freeman and Company, New York(1990), each incorporated herein by reference. One modality of genetherapy involves (a) obtaining from a patient a viable sample of primarycells of a particular cell type; (b) inserting into these primary cellsa nucleic acid segment encoding a desired gene product; (c) identifyingand isolating cells and cell lines that express the gene product; (d)re-introducing cells that express the gene product; (e) removing fromthe patient an aliquot of tissue including cells resulting from step cand their progeny; and (f) determining the quantity of the cellsresulting from step c and their progeny, in said aliquot. Theintroduction into cells in step (b) of a vector that encodes a sequence(for a “desired gene product”) which will block mesothelin expression oractivity can be useful in inhibiting or slowing the growth of tumorcells associated with mesothelin.

V. Vaccine Development

Vaccine Development Using Mesothelin Amino Acid Sequence.

Substances suitable for use as vaccines for the prevention of andinhibition of the growth of tumors bearing mesothelin and methods foradministering them may be employed. The vaccines are directed againstmesothelin. Preferably, the vaccines comprise mesothelin derivedantigen.

Vaccines can be made recombinantly. Typically, a vaccine will includefrom about 1 to about 50 micrograms of antigen or antigenic protein orpeptide. More preferably, the amount of protein is from about 15 toabout 45 micrograms. Typically, the vaccine is formulated so that a doseincludes about 0.5 milliliters. The vaccine may be administered by anyroute known in the art. Preferably, the route is intraperitoneally orparenteral.

There are a number of strategies for amplifying an antigen'seffectiveness, particularly as related to the art of vaccines. Forexample, cyclization or circularization of a peptide can increase thepeptide's antigenic and immunogenic potency. See U.S. Pat. No. 5,001,049which is incorporated by reference herein. More conventionally, anantigen can be conjugated to a suitable carrier, usually a proteinmolecule. This procedure has several facets. It can allow multiplecopies of an antigen, such as a peptide, to be conjugated to a singlelarger carrier molecule. Additionally, the carrier may possessproperties which facilitate transport, binding, absorption or transferof the antigen.

For parenteral administration, examples of suitable carriers are thetetanus toxoid, the diphtheria toxoid, serum albumin and lamprey, orkeyhole limpet, hemocyanin because they provide the resultant conjugatewith minimum genetic restriction. Conjugates including these universalcarriers can function as T cell clone activators in individuals havingvery different gene sets.

The conjugation between a peptide and a carrier can be accomplishedusing one of the methods known in the art. Specifically, the conjugationcan use bifunctional cross-linkers as binding agents as detailed, forexample, by Means and Feeney, “A recent review of protein modificationtechniques,” Bioconjugate Chem. 1:2-12 (1990).

The antigen may be combined or mixed with various solutions and othercompounds as is known in the art. For example, it may be administered inwater, saline or buffered vehicles with or without various adjuvants orimmunodiluting agents. Examples of such adjuvants or agents includealuminum hydroxide, aluminum phosphate, aluminum potassium sulfate(alum), beryllium sulfate, silica, kaolin, carbon, water-in-oilemulsions, oil-in-water emulsions, muramyl dipeptide, bacterialendotoxin, lipid X, Corynebacterium parvum (Propionibacterium acnes),Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin,lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran,blocked copolymers or other synthetic adjuvants. Such adjuvants areavailable commercially from various sources, for example, Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.) or Freund's IncompleteAdjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.).Other suitable adjuvants are Amphigen (oil-in-water), Alhydrogel(aluminum hydroxide), or a mixture of Amphigen and Alhydrogel. Onlyaluminum is approved for human use.

The proportion of antigen and adjuvant can be varied over a broad rangeso long as both are present in effective amounts. For example, aluminumhydroxide can be present in an amount of about 0.5% of the vaccinemixture (Al₂O₃ basis). On a per-dose basis, the amount of the antigencan range from about 0.1 μg to about 100 μg protein per patient. Apreferable range is from about 1 μg to about 50 μg per dose. A morepreferred range is about 15 μg to about 45 μg. A suitable dose size isabout 0.5 ml. After formulation, the vaccine may be incorporated into asterile container which is then sealed and stored at a low temperature,for example 4° C., or it may be freeze-dried. Lyophilization permitslong-term storage in a stabilized form.

The treatment may consist of a single dose of vaccine or a plurality ofdoses over a period of time. It is preferred that the doses be given toa patient suspected of having mesothelin bearing tumor cells. Theantigen of the invention can be combined with appropriate doses ofcompounds including influenza antigens, such as influenza type Aantigens. Also, the antigen could be a component of a recombinantvaccine which could be adaptable for oral administration.

Vaccines of the invention may be combined with other vaccines for otherdiseases to produce multivalent vaccines. A pharmaceutically effectiveamount of the antigen can be employed with a pharmaceutically acceptablecarrier such as a protein or diluent useful for the vaccination ofmammals, particularly humans. Other vaccines may be prepared accordingto methods well-known to those skilled in the art.

Those of skill will readily recognize that it is only necessary toexpose a mammal to appropriate epitopes in order to elicit effectiveimmunoprotection. The epitopes are typically segments of amino acidswhich are a small portion of the whole protein. Using recombinantgenetics, it is routine to alter a natural protein's primary structureto create derivatives embracing epitopes that are identical to orsubstantially the same as (immunologically equivalent to) the naturallyoccurring epitopes. Such derivatives may include peptide fragments,amino acid substitutions, amino acid deletions and amino acid additionswithin the amino acid sequence for mesothelin. For example, it is knownin the protein art that certain amino acid residues can be substitutedwith amino acids of similar size and polarity without an undue effectupon the biological activity of the protein.

Using the mesothelin amino acid sequence information, one of skill inthe art can perform epitope mapping against sera isolated from patientswith ovarian cancers or mesotheliomas. Relatively strong epitopes may beidentified and common epitope(s) may also be recognized. The epitopemapping against human sera can also be extended to a screening ofepitope-peptides against activated human lymphocytes in order toidentify potential T-cell epitopes. Theoretically, it is not likely thatT-cell epitopes of mesothelin will be found in human T-cells, butmutations induced in mesothelin may create new epitopes which may berecognized by T-cells. Mutant mesothelin can easily be generatedrandomly using a phage display method. The resultant library is screenedby human sera from patients suffering from malignant mesothelioma andovarian cancer. Thus, suitable antigenic peptides may be identified formesothelin-derived vaccines.

VI. Kits.

This invention further embraces diagnostic kits for detecting for thepresence of mesothelin in tissue samples or in serum, comprising acontainer having a nucleic acid or an antibody or other targeting agentspecific for mesothelin and instructional material for the detection ofmesothelin.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference.

VI. Models for Evaluation of Therapies Directed to Mesothelin.

The mesothelin cDNA can be transfected into established tumor cell lineswhere it will express the protein. The transfected cell lines can beused to grow tumors in mice or other mammals to provide a model fortesting therapies directed to controlling, suppressing or regulatingmesothelin expression. Transfected tumor cell lines can be transplantedinto the test mammal. The mammal can then be subjected to a drug ofinterest and subsequent tumor cell activity can be monitored todetermine whether the drug of interest has anti-tumor effects. Tumorcell lines that have been found to be particularly good candidates forthis procedure include, mouse NIH 3T3 cells (tumorigenic cell lines),A431 human ovarian tumor cells and MCF-7 breast tumor cells, A2780 humanovarian tumor cells and OVCAR-3 human epidermoid carcinoma cells.

EXAMPLES A. Materials and Methods

1. Cells and antibodies. Human ovarian tumor cell line, OVCAR-3, andcell lines A431, KB, MCF-7, COS-1, WI-38 and NIH 3T3 were obtained fromthe American Type Culture Collections (ATCC, Rockville, Md.). Cells werecultured either in RPMI 1640 or DMEM media (GIBCO Laboratories, GrandIsland, N.Y.), supplemented with L-glutamine (2 mM), penicillin (50μg/ml), streptomycin (50 units/ml) and 5-10% fetal bovine serum (GIBCO).NIH 3T3 transfectants were grown in DMEM with 0.8 mg/ml of G418 (GIBCO).Cells were used when they reached 80-90% confluency after washing threetimes with ice-cold PBS (GIBCO). MAb K1 and antibody MOPC-21 have beendescribed (Chang, K., et al., Int. J. Cancer 50, 373-381 (1992)) andwere used in a concentration of 5-10 μg/ml.

2. Isolation of the cDNA clones. The HeLa S3 cDNA library (ClonTech,Palo Alto, Calif.) was screened at approximately 50,000 pfu/150 mmfilter as described previously (Chang, K., and Pastan, I., Int. J.Cancer 57, 90-97 (1994)) using protein A-purified MAb K1 (5 μg/ml) andperoxidase-conjugated goat anti-mouse IgG (H+L) (10 μg/ml, JacksonImmunoResearch Lab, Inc., West Grove, Pa.). Two positive plaques (λ6-1,λ6-2) were isolated and the phages were purified to homogeneity by threeor more rounds of screening. After verification of their specificitywith MAb K1 by showing they did not react with a control MOPC-21antibody, single-plaque isolates of λ6-1 and λ6-2 were used to prepare 5to 10 phage-plates, followed by extraction and purification of phage DNAwith a lambda phage DNA kit (Qiagen, Inc., Chatsworth, Calif.). PhageDNA was then digested with EcoRI and the insert subcloned into the EcoRIsite of a pcDNAI/Amp (Invitrogen Corporation, San Diego, Calif.) vectorusing a rapid ligation protocol (Chang, K., and Pastan, I., Int. J.Cancer 57, 90-97 (1994)). Plasmid DNAs were isolated using Qiagene'splasmid DNA isolation kit (Chang, K., and Pastan, I., Int. J. Cancer 57,90-97 (1994)). Restriction mapping using XhoI, EcoRI, SalI, BamHI, NcoI,and DNA sequencing revealed that the two plasmid clones (p6-1 and p6-2)had identical 1500 base-pair inserts.

To isolate a longer clone, the insert of p6-1 was purified to make acDNA probe (specific activity=8.5×10⁵ cpm/ml) by random priming. TheHeLa S3 cDNA library was re-screened using the filter hybridizationmethod described previously (Chang, K., and Pastan, I., Int. J. Cancer57, 90-97 (1994)). 14 lambda clones were isolated and purified, andtheir insert sizes were assessed by digestion with EcoRI. Four largeinserts were subcloned into a pcDNAI/Amp plasmid vector (p9, p13-1, p16and p18-1). p9 contained the largest insert with a long open readingframe.

3. DNA sequencing analysis. Using T3 and T7 promoter primers and twenty17 bp synthetic primers, the entire cDNA insert of p9 was sequencedusing the method described by Sanger (Sanger, F., et al., Proc. Natl.Acad. Sci. USA 74, 5463-5467 (1977)) and an automatic cycle sequencingmethod.

4. Northern blot analysis. Total RNAs (20 μg) from OVCAR-3, KB, MCF-7,A431 and WI38 were electrophoresed on a 1% agarose gel in MOPS bufferwith 16.6% formaldehyde, and then transferred to a Nylon paper. Northernhybridization was done with a method described before (Chang, K., andPastan, I., Int. J. Cancer 57, 90-97 (1994)). The blot washed andreprobed with a ³²P-labeled human β-actin cDNA as an internal control toassess the integrity and quantity of the RNA samples loaded.

5. In Vitro transcription and translation. TNT Coupled ReticulocyteLysate System, canine pancreatic microsomal membrane, 2 μg of plasmidDNAs of p9 (pcDICAK1-9), pAPK1 (Chang, K., and Pastan, I., Int. J.Cancer 57, 90-97 (1994)), to eliminate and ³H leucine were used in an invitro transcription/translation and translocation/processing experimentaccording to the protocol of the manufacturer (Promega, Madison, Wis.,USA). Translation products were resolved on a 10% SDS-PAGE reducing gel.The proteins were fixed and the unincorporated label was removed bysoaking the gel three times in 200 ml of buffer, 40% methanol and 10%acetic acid in deionized water for 30 min. The gels were then soaked for30 min in 200 ml of INTENSIFY Part A and Part B (NEN Research Product,Boston, Mass.). After drying, the translated products were visualized byautoradiography.

6. Expression of the cloned cDNAs in mammalian cells. Transienttransfections of COS cells were performed using pcDICAK1-9 (p9) andLipofectAMINE (GIBCO) following the manufacturer's protocol (GIBCO).COS1 cells were plated a day before the experiment at 2.5×10⁵ cells/60mm dish. 24 μl of LipofectAMINE and 76 μl of OptiMEMI medium were mixedwith 10 μg of pcDNAI/Amp vector, or pcDICAK1-9 in 100 μl of OptiMEMImedium at room temperature for 30 min. After washing the COS-1 cellswith OptiMEMI twice, 2.4 ml OptiMEMI were added to the transfectionmixtures and overlaid onto COS1 cells, followed by incubation at 37° C.for 5 hours. 2.6 ml of DMEM with 20% FBS were then added into each dish.48 hours after transfection, the dishes were subjected toimmunofluorescence labeling as described (Chang, K., et al., Int. J.Cancer 50, 373-381 (1992); Chang, K., et al., Cancer Res. 52, 181-186(1992)) or other treatments. The insert from plasmid p9 (in pcDNAI/Amp)was also subcloned into a pcDNA3 (Invitrogen) vector for stabletransfection. Plasmid minipreps were made using Qiagen's MiniprepPlasmid DNA Kit and orientation of the insert in individual clone wasdetermined by restriction mapping. The resulting plasmid, pcD3CAK1-9,was then used to transfect NIH 3T3, MCF-7, A431 and OVCAR-3 cells byDNA-calcium phosphate precipitation as described (Chen, C. and Okayama,H., Mol. Cell. Biol. 7, 2745-2752 (1987)). After overnight exposure tothe precipitate, the cells were washed with PBS three times and fed withfresh DMEM/10% FBS medium for 2-3 days. Geneticin G418 sulfate (0.8mg/ml) was added and the cultures were maintained until colonies of 2-3mm in diameter were formed. Colonies were then transferred into wells ofa 96 well plate and then into a 35 mm dish when they were 80% confluent.Transfected cells were screened by immunofluorescence (Chang, K., etal., Int. J. Cancer 50, 373-381 (1992); Chang, K., et al., Cancer Res.52, 181-186 (1992)) and positive cells were further subcloned by limiteddilution as described (Chang, K., et al., Int. J. Cancer 50, 373-381(1992)). One of the NIH 3T3 transfectant clones, NIH 3T3 K20, was chosenfor further study. To localize the expression of CAK1, both cell surfaceand intracellular immunofluorescence labeling was also performedaccording to methods described before (Chang, K., et al., Cancer Res.52, 181-186 (1992)).

7. Treatment of the transfected cells with PI-PLC. CAK1 cDNA transfectedNIH 3T3 cells (NIH 3T3 K20) were grown in 175 mm² flasks, and when theyreached 90% confluency, the cells were washed in PBS for three times.The cells were incubated with either 5 ml of 1.25 U/ml PI-PLC (fromBacillus cereus; Boehringer Mannheim Biochemicals) or 0.05%trypsin/0.052 mM EDTA for 30 min at 37° C. and 30 min at roomtemperature with shaking. The supernatants were collected and aftercentrifugation at 1000×g and concentrated about 10 fold using Centricon30 (Amicon, Inc., Beverly, Mass.). The concentrated supernatants wereused in SDS-PAGE and immunoblot analysis. The enzyme-treated cells canbe recultured and the recovery of CAK1 expression can be seen afterovernight culture. Treatment with PI-PLC was done in a similar mannerusing 35 mm diameter dishes followed by immunofluorescence labeling ofthe treated cells (Chang, K., et al., Cancer Res. 52, 181-186 (1992)).

8. Immunoblotting analysis of the transfected NIH 3T3 cells. Membraneand cytosolic fractions from transfected NIH 3T3 K20 cells (Chang, K.,and Pastan, I., Int. J. Cancer 57, 90-97 (1994)) were subjected to 12.5%SDS-PAGE and the resolved proteins were transferred to nitrocellulose.Immunoblotting was performed as previously described (Chang, K., et al.,Int. J. Cancer 51, 548-554 (1992); Chang, K., and Pastan, I., Int. J.Cancer 57, 90-97 (1994)).

B. Results

Expression cloning was used to isolate the CAK1 cDNA. We previouslyobserved that MAb K1 reacts with OVCAR-3 and HeLa cells. Because we wereunable to isolate the cDNA from an OVCAR-3 library (Chang, K., andPastan, I., Int. J. Cancer 57, 90-97 (1994)), we screened a HeLa cDNAlibrary expressed in λgt11 as described above. A total of 1×10⁶ phageswere screened and two phage clones (λ6-1 and λ6-2) were identified. DNAsequencing showed both phages contained the same 1.5 kb insert. Theinsert hybridized to mRNA from OVCAR-3 and KB cells (a HeLa subclonewhich also reacts with MAb K1) but not to RNA from many other cell linesindicating that the cDNA is specific for cells reacting with MAb K1. 20μg of total RNA from OVCAR-3 cells (lane 1), MCF-7 cells (lane 2), KBcells (a HeLa subclone, lane 3), A431 cells (lane 4) and W138 cells(lane 5) were resolved by electrophoresis transferred to nylon paper andblotted with a ³²P-labeled CAK1 probe. Hybridization with an actin probeshowed that the lanes were equally loaded. The mRNA detected is 2.2 kbin size indicating that the insert isolated was not full length. Theinsert contained an open reading frame, a stop codon and a poly A tailbut the 5′ end appeared to be missing. Therefore, the phage library wasrescreened with one of the inserts and 14 new phages with cDNA insertsof various sizes isolated. The largest insert (#9) was 2138 bp long andwhen sequenced contained an open reading frame of 1884 bp (FIG. 1). Itcontains a typical Kozak sequence (Kozak, M., Nucleic Acids Res. 5,8125-8148 (1987)) (AXXATGG) followed by an open reading frame thatencodes a 69 kD protein. The sequence was not present in various databases examined (EMBL-GenBank). Because the CAK1 antigen was originallyfound to be about 40 kD in size, several experiments were carried out todetermine if clone 9 encoded CAK1.

1. In vitro translation. Insert 9 was cloned into a pcDNAI/Amp vector tomake pcDICAK1-9 and used in the TNT reticulocyte system. pcDICAK1-9plasmid DNA (lanes 1 and 2), and pcDIAPK1 (lanes 3 and 4) were used in aTNT coupled reticulocyte lysate system in the presence (+) or absence(−) of pancreatic microsomal membrane (m). The products were resolved ona 10% reducing SDS-PAGE and autoradiographed. A 69 kD protein wasproduced. In the presence of pancreatic microsomes (lane 2), a slightlylarger protein was observed indicating the protein had been insertedinto microsomes and glycosylated. As a control, a cDNA encoding a 30 kDcytosolic protein that also reacts with MAb K1 (Chang, K., and Pastan,I., Int. J. Cancer 57, 90-97 (1994)) was subjected to the same analysis.The size of the protein was unaffected by the presence of microsomes.

2. Expression in cultured cells. pcDICAK1-9 was transfected into COScells for transient expression. pcDNAI/Amp vectors with insert 9 orwithout insert were transfected into COS cells. Two days later, thecells were immunocytochemically labeled with MAb K1 at 4° C. (forsurface labeling) or at 23° C. (for intracellular labeling) andphotographed (Magnification ×250). The specific labeling pattern of COScells transfected with insert 9 using MAb K1 was observed. Innonpermeabilized cells, a typical cell surface fluorescent pattern isdetected. In permeabilized cells, strong staining of the Golgi region isevident. No cytosolic staining was detected. Also, no immunoreactivitywas detected in cells transfected with vector without insert or controlinserts. Thus, insert 9 encodes a cell surface protein that is alsopresent in the Golgi.

3. Size and processing of CAK1 antigen. To determine the size of theantigen produced by cells transfected with insert 9, NIH 3T3 cells weretransfected with pcD3CAK1-9 to make stable cell lines. Stablytransfected clones were produced as described above and the presence ofantigen on the surface was confirmed by immunofluorescence. Thenmembrane and cytosolic fractions were prepared from NIH 3T3 K20 cellsand from OVCAR-3 cells, subjected to SDS-PAGE and analyzed byimmunoblotting with MAb K1. Approximately 100 μg of membrane fraction(lanes 1 and 3) or cytosolic fraction (lanes 2 and 4) of the transfectedNIH 3T3 (pcD3CAK1) and mock control (pcD3) and membrane (lane 5) orcytosolic fraction (lane 6) of OVCAR-3 cells were electrophoresed andimmunoblotted with MAb K1. As previously reported, the major reactivityin OVCAR-3 cells is with a doublet of about 40 and 43 kD that is presentin membranes but not in the cytosol. In the transfectants, two bands ofequal intensity were detected in the membrane fraction; one of about 40kD and a second of about 71 kD. No signal was detected in the cytosol.These data suggest that CAK1 is made as a large molecular weightprecursor that is processed by proteolysis to an approximately 40 kDform.

4. Nature of cell surface attachment. To determine if CAK1 was attachedto the transfectants via a PI linkage as it is in OVCAR-3 cells (Chang,K., et al., Cancer Res. 52, 181-186 (1992)), the NIH 3T3 transfectantcell line k20 was treated with PI-PLC for 60 min. The transfected NIH3T3 k20 cells were treated with PI-PLC and labeled with MAb K1 asdescribed above. The CAK1 signal was completely abolished after PI-PLCtreatment. A strong cell surface labeling pattern was observed inuntreated cells. Fluorescence was absent after treatment with PI-PLC. Inphase contrast images before (B) and after (D) treatment, the treatedcells are still attached to the dish but are slightly altered in shape.The medium from PI-PLC treated cells was concentrated, subjected toSDS-PAGE and analyzed with MAb K1. A band of about 70kD was detected,but no lower molecular weight bands were detected.

C. Summary of Results

Thus, the above describes the molecular cloning of the CAK1 antigenwhich is found on mesothelium, mesotheliomas, ovarian cancers and somesquamous cell carcinomas. We have designated this antigen mesothelin toreflect its presence on mesothelial cells. One unexpected feature ofmesothelin is that its cDNA encodes a 69 kD protein, whereas the antigenpresent on OVCAR-3 cells, used to isolate MAb K1, has a molecular weightof 40,000 Daltons. The DNA sequence and the deduced amino acid sequenceof CAK1 is shown in FIG. 1. The cDNA is 2138 bp in length and containsan open reading frame of 1884 bp. The protein it encodes contains 628amino acids with a calculated molecular weight of 69001 daltons. Ahomology analysis was performed with nucleotide or amino acid sequencesand none was detected using EMBL-GenBank accessed by the GCG program.The protein contains four potential N-linked glycosylation sites N-X-Sor N-X-T that are shown in bold letters. A typical signal sequence isnot present at the amino terminus. However, a short hydrophobic segmentis located 15 amino acids from the first methionine (FIG. 1). Thissequence might function as a signal sequence for membrane insertion,because the protein is found on the cell surface and is inserted intomicrosomes during cell free translation. Also present is a putativeproteolytic processing site, RPRFRR (SEQ ID NO:8), beginning at aminoacid 293 (FIG. 1). This site is recognized by furin, a proteaseimportant in the processing of several membrane proteins as well as inthe activation of Pseudomonas and diphtheria toxins (Chiron, M. F., etal., J.B.C. 269(27):18169-18176 (1994)). The 40 kD form appears to bederived from a 69 kD precursor by several processing steps. These aresummarized in FIG. 2. Initially, mesothelin is made as a 69 kDpolypeptide with a hydrophobic tail which is probably removed andreplaced by phosphatidylinositol (Chang, K., et al., Cancer Res. 52,181-186 (1992)). After glycosylation at one or more of its four putativeN-linked glycosylation sites, it is cleaved by a protease to yield the40 kD fragment (or doublet) found on the surface of OVCAR-3 cells and asmaller (˜31 kD) fragment. The latter could be released into the mediumand/or further degraded. The amino terminal fragment has recently beendetected in the medium of OVCAR-3 cells (our data). In transfected NIH3T3 and MCF-7 cells, we find approximately equal amounts of 70 kD and 40kD proteins. We originally detected the 40 kD form in OVCAR-3 and HeLacells and did not notice a larger form. Reexamination of the OVCAR-3 andHeLa cell gels reveals a trace amount of the 70 kD precursor.

1. An isolated protein comprising the full-length amino acid sequence ofSEQ ID NO:2.
 2. A composition comprising an isolated protein comprisingthe full-length amino acid sequence of SEQ ID NO:2, and apharmaceutically acceptable carrier.
 3. A composition of claim 2,further comprising an adjuvant.